The Bowman-Birk proteolytic enzyme inhibitor (BBI) is a designation of a family of stable low molecular weight trypsin and chymotrypsin enzyme inhibitors found in soybeans and various other seeds, mainly leguminous seeds, and vegetable materials. The Bowman-Birk enzyme inhibitor was first noted by Bowman (Proc. Soc. Exptl. Med. 1946, 63, 574) and subsequently purified and characterized by Birk, Y. (Biochim. Biophys. Acta 1961, 54, 378-381) and Birk. Y. et al, (1968) Biochemical Preparations Vol.12, 25-29 (W.E.M. Lands, Ed.) John Wiley & Sons Inc., later to be known and referred to as the Bowman-Birk Inhibitor (BBI), (hereinafter designated "BBI"). The BBI was reviewed by Kobayashi et al., J. Biochem. 1982, 91, 1511; by Birk, Int. J. Peptide Protein Res. 1985, 25, 113; and by Birk, pp. 257 in Hydrolytic Enzymes, Neuberger and Brocklehurst (Eds.), Elsevier Science Publications B.V., Amsterdam, 1987. The BBI is a protein characterized by its low molecular weight of .sup..about. 8000 (in non-associated monomers), high concentration (.sup..about. 20%) of cystine, high solubility, resistance to heat denaturation and having the capacity to inhibit trypsin and chymotrypsin at independent inhibitory sites.
It is known that both crude and purified BBI prevent, or reduce various types of induced malignant transformation of cells in culture and experimental animals. There are also known various methods of obtaining crude and purified BBI products. A review of the relevant literature is provided by Kennedy et al. in U.S. Pat. No. 5,338,547, which is hereby incorporated in its entirety.
In view of the possibility that a BBI product may provide a potential remedy for prevention and amelioration of carcinogenesis, attempts have been made to prepare pure and sundry BBI preparations as potential therapeutic medicaments for diverse cancer conditions by various methods (U.S. Pat. No. 5,217,717 and U.S. Pat. No. 5,338,547). Preparing pure BBI, however, involves costly techniques.
It is now generally accepted that the responsibility for the suppression or inhibition of carcinogenesis, or the malignant transformation of cells from normal cells to cancer cells by BBI is due mainly to the chymotrypsin inhibitory site of the BBI molecule and not to the trypsin inhibitory site, (Yavelow et al., Proc. Natl. Acad. Sci. USA 1985, 82, 5395; Kennedy, Carcinogenesis 1985, 6, 1441; and Kennedy, pp. 9 in Protease Inhibitors as Cancer Chemopreventive Agents, Troll and Kennedy Eds.!, Plenum Press, New York, 1993).
The above mentioned U.S. Pat. No. 5,338,547, discloses a method for suppressing and inhibiting carcinogenesis with highly active BBI concentrate products wherein the level of biological activity is measured by chymotrypsin inhibitor content. These BBI concentrate products are made from acidic soybean solubles obtained from defatted soybean flour or flakes which were extracted with aqueous acid at pH 4 to 5, and from which the insolubles were removed by centrifugation. The soybean solubles were subjected to ultrafiltration to produce a crude BBI concentrate, which was diluted and spray dried to produce the final dried BBI concentrate product. In a preferred process embodiment disclosed in this patent the crude BBI concentrate is treated with acetone to produce a BBI concentrate precipitate which is air dried, ground, reslurried with water, filtered and then lyophilized or spray dried to produce the final BBI concentrate product. This product is stated to be an improved inhibitor of carcinogenesis. Kennedy et al. also mention that the BBI concentrate product can be further purified, by a method described by Odani et al. (J. Biochem. 1973, 74, 857), which method involves fragmenting the BBI product into two separated fragments, one fragment having the trypsin inhibitory site and the other fragment having the chymotrypsin inhibitory site. The inhibiting activity of the fraction having the chymotrypsin inhibitory site was, however, severely impaired.
It is known that ingesting products having a trypsin inhibitory effect such as the Kunitz Inhibitor and the BBI can have considerable deleterious consequences. These products can cause enlargement of the pancreas and increase of pancreatic proteolytic activity. On the other hand, the chymotrypsin inhibitory site on the BBI molecule does not cause pancreatic hypertrophy and has no significant effect on the amount of pancreatic proteinases (Pfeifer et al., J. Mol. Cell. Cardiol. 1981, 12, 37). This was demonstrated by Madar et al., Comp. Biochem. Physiol. 1974 48B, 251, by blocking the trypsin inhibitory site and comparing the blocked product with an unblocked product for pancreatic hypertrophy. Various blocking methods were used by Madar et al. Among these were the maleylation and succinylation of the BBI with maleic and succinic anhydride respectively.